DNA replication is regulated by polymerases and several other proteins that act on DNA in association with the bacterial membrane. These interactions are altered by the peptide antibiotic phleomycin. Phleomycin arrests normal DNA replication by causing discrete breaks in cellular DNA and by releasing the DNA from its association with the bacterial membrane. Conversely, phleomycin enhances DNA synthesis in toluenized cells. We will validate the preliminary observation that phleomycin-treated DNA in toluenized cells has enhanced template activity primarily for polymerase II by testing the template capacity of purified phleomycin-treated DNA with isolated polymerases I, II and III, in vitro. We will determine the nature of the phleomycin induced changes in DNA that enhance template activity, and we will relate these changes to alterations in the normal DNA-membrane associations. We will use standard methods of DNA polymerase isolation and assay. We will use density gradient, and polyacrylamide gel electrophoresis analyses to identify changes in the DNA and in the membranes.